Therapeutic ribonucleases

ABSTRACT

The present invention relates to the use of ribonucleases (RNases) in the treatment or prevention of disease.

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of allowed U.S. patentapplication Ser. No. 13/231,050, filed Sep. 13, 2011, which iscontinuation of U.S. Pat. No. 8,029,782, issued Oct. 4, 2011, whichclaims priority to expired U.S. Provisional Patent Application Ser. No.61/101,905, filed Oct. 1, 2008, each of which is herein incorporated byreference in its entirety.

FIELD OF THE INVENTION

The present invention relates to the use of ribonucleases (RNases) inthe treatment or prevention of disease.

BACKGROUND OF THE INVENTION

In the U.S. population, mortality associated with the 15 most commoncancer types alone has been estimated to approach 170 deaths annuallyper 100,000 individuals (Martin L Brown, Joseph Lipscomb and ClaireSnyder, 2001, THE BURDEN OF ILLNESS OF CANCER: Economic Cost and Qualityof Life, Ann. Rev. Public Health, 22: 91-113). Currently, there are anestimated 1,437,180 new cases of cancer and 565,650 deaths each year(American Cancer Society Cancer Facts and Figures 2008). The economicburden of cancer has been estimated to exceed $96B in 1990 dollars(Brown et al, 2001).

The term “chemotherapy” simply means the treatment of disease withchemical substances. The father of chemotherapy, Paul Ehrlich, imaginedthe perfect chemotherapeutic as a “magic bullet;” such a compound wouldkill invading organisms or cells without harming the host. Whilesignificant progress has been made in identifying compounds that kill orinhibit cancer cells and in identifying methods of directing suchcompounds to the intended target cells, the art remains in need ofimproved therapeutic compounds.

There are a range of different types of chemotherapeutic agentsavailable, including small molecules and biologics such as nucleic acidcompounds, polypeptide compounds, or derivatives thereof. In general,properties of chemotherapeutics requiring consideration includeefficacy, pharmacokinetic properties, and ease of manufacture. Proteintherapeutics in general may offer particular advantages as alternativesto small molecules; however, effective protein drugs must have a balanceof optimal properties that include specificity, cytotoxicity, affinityfor their target, safety, solubility, amenability to effective delivery,stability, and longevity in the body (clearing time). A candidate drughaving superiority in any one of these properties may not possess theoptimal balance of features overall to serve as an effective drug.

There is need in the art for additional anti-cancer chemotherapeuticsthat have an effective complement of features for use in therapeuticsettings.

SUMMARY OF THE INVENTION

The present invention relates to the use of RNases in the treatment orprevention of disease. In some embodiments, RNases are used for thetreatment or prevention of human disease such as cancer. In someembodiments, a variant of recombinant human RNase 1 has an optimalbalance of desirable properties for the treatment or prevention of humancancer. Such properties include but are not limited to stability,cytotoxicity towards pathogenic cells, efficacy of degradation ofpathogenic RNA of any origin including viral RNA, evasion of binding byRNase inhibitors, resistance to degradation by proteases, delivery totarget cells, efficiency of import into the cell, dose responseproperties, pharmacokinetic properties, and longevity within the humanbody. In some embodiments, therapeutic compositions and methods of theinvention employ a variant of human RNase 1 that has amino acid changescompared to a wild type enzyme: R4C, G38R, R39G, N67R, G89R, S90R, andV118C. In some embodiments, the ribonuclease comprises or consists ofSEQ ID NO:1:

KESCAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQRGCKPVNTFVHEPLVDVQNVCFQEKVTCKRGQGNCYKSNSSMHITDCRLTNRRRYPNCAYRTSPKERHIIVACEGSPYVPCHFDASVEDST.

In some embodiments, the amino acid sequence comprises or consists ofSEQ ID NO:1 having an N-terminal methionine (SEQ ID NO:4):

MKESCAKKFQRQHMDSDSSPSSSSTYCNQMMRRRNMTQRGCKPVNTFVHEPLVDVQNVCFQEKVTCKRGQGNCYKSNSSMHITDCRLTNRRRYPNCAYRTSPKERHIIVACEGSPYVPCHFDASVEDST.

In some embodiments, the ribonuclease is recombinantly produced. In someembodiments, the ribonuclease is encoded by a nucleic acid moleculecomprising SEQ ID NO:2:

AAA GAA TCT TGC GCT AAA AAA TTC CAG CGT CAG CAC ATG GAC TCT GACTCT TCT CCG TCT TCT TCT TCT ACT TAC TGC AAC CAG ATG ATG CGT CGCCGT AAC ATG ACT CAG CGT GGT TGC AAA CCG GTT AAC ACT TTC GTT CATGAA CCG CTG GTT GAC GTT CAG AAC GTT TGC TTC CAG GAA AAA GTT ACTTGC AAA CGC GGT CAG GGT AAC TGC TAC AAA TCT AAC TCT TCT ATG CATATC ACT GAC TGC CGT CTG ACG AAT CGT CGC CGT TAC CCG AAC TGC GCTTAC CGT ACT TCT CCG AAA GAA CGT CAT ATC ATC GTT GCT TGC GAA GGTTCT CCG TAC GTT CCG TGT CAT TTC GAC GCT TCT GTT GAA GAC TCT ACC

In some embodiments, an expression vector encoding the ribonucleasefurther comprises a leader sequences, such as SEQ ID NO:3:

TT GNT TAA CTT TAA GAA GGA GAT ATA CATATG

In some embodiments, an expression vector encoding the ribonucleasefurther comprises one or more stop codons (e.g., TAA and/or TGA).

In some embodiments, the RNase is conjugated to water-soluble moietiesas a means of providing improved physical properties. In someembodiments, the water-soluble moiety is a polyethylene glycol (PEG)molecule. In some embodiments, the RNase is conjugated or fused to, orotherwise associated with, a moiety that targets the RNase to a specificcell type, such as a diseased or cancerous cell.

Thus, in some embodiments, the present invention provides a composition(e.g., a therapeutic preparation) comprising a ribonuclease (e.g., apurified ribonuclease) having a human RNase 1 sequence with amino acidmodifications: R4C, G38R, R39G, N67R, G89R, S90R, and V118C (e.g.,comprising or consisting of SEQ ID NO:1). In some embodiments, thepresent invention provides an expression vector comprising a nucleicacid sequence encoding such a ribonuclease (e.g., comprising SEQ IDNO:2). In some embodiments, the present invention provides a host cellcomprising such an expression vector. In some embodiments, the presentinvention further provides methods of treating subjects (e.g., humansubject suffering from or suspected of suffering from cancer) byadministering a ribonuclease, alone or in combination with other agents,to the subject. The administration may be at one or more time points ina therapeutically effective dose (e.g., administered at 0.01 to 100mg/kg body weight of the subject per week for one or more weeks; per dayfor one or more days; or per treatment for one or more treatments).

As used herein, the term “variant of a ribonuclease” refers to amodified ribonuclease that retains enzymatic activities similar to that(e.g., that has at least 60%, at least 75%, at least 85%, at least 95%,or at least 99% of activity retained) associated with the non-modifiedribonuclease from which it was derived. Tests for measuring enzymaticactivities are described herein and are known in the art. The variantmay be a variant of a natural enzyme or may be a variant of anon-natural enzyme. For example, a particular non-natural syntheticribonuclease having SEQ ID NO:1 may be modified to include one or moreamino acid changes that result in a variant of SEQ ID NO:1. In somepreferred embodiments, the variant has one or a limited number of aminoacid substitutions (e.g., conservative or non-conservativesubstitutions), additions, or deletions (e.g., truncations) compared tothe non-modified ribonuclease.

As used herein, the term “variant of a ribonuclease retaining RNAdegradation activity” refers to a variant of ribonuclease (e.g., SEQ IDNO:1) that has at least 50%, at least 60%, at least 75%, at least 85%,at least 95%, or at least 99% of RNA degradation activity of thenon-modified ribonuclease from which it was derived. RNA degradationactivity may be measured using any suitable assay including, but notlimited to, visualization and quantitation of a degraded RNA sampleusing agarose or polyacrylamide gel electrophoresis. In some preferredembodiments, the variant has one or a limited number of amino acidsubstitutions (e.g., conservative or non-conservative substitutions),additions, or deletions (e.g., truncations) compared to the non-modifiedribonuclease.

As used herein, the term “variant of a ribonuclease having substantiallythe same cell killing activity, cytotoxic activity, cytostatic activity,or cell damaging activity” refers to a variant of a ribonuclease (e.g.,SEQ ID NO:1) that has at least 50%, at least 60%, at least 75%, at least85%, at least 95%, or at least 99% of the cell killing activity,cytotoxic activity, cytostatic activity, or cell damaging activity ofthe non-modified ribonuclease from which it was derived. For example, insome embodiments, the activity is the ability to kill or otherwiseaffect cancer cells. In other embodiments, it is the ability to reducetumor size in animals. In yet other embodiments, the activity is theability to reduce symptoms of a disease characterized by aberrant cellgrowth (e.g., cancer). Activity may be measured using any suitablemethod including, but not limited to, commercially available cellviability assays, measurement of tumor size, and commercially availablecell proliferation assays. In some preferred embodiments, the varianthas one or a limited number of amino acid substitutions (e.g.,conservative or non-conservative substitutions), additions, or deletions(e.g., truncations) compared to wild type enzyme.

As used herein, the term “variant of a ribonuclease retaining proteinfolding properties” refers to a variant of a ribonuclease (e.g., SEQ IDNO:1) that exhibits similar protein folding properties as thenon-modified ribonuclease from which it was derived. Protein foldingproperties include speed of protein folding and folding of properstructure (folding that substantially retains the activity of thenon-modified ribonuclease from which it was derived). In preferredembodiments variants fold with at least 50%, at least 60%, at least 75%,at least 85%, at least 95%, or at least 99% or more of the speed of thenon-modified ribonuclease from which it was derived. Assays for proteinfolding are well known in the art and include, but are not limited to,spectroscopic and enzymatic (e.g. RNA degradation) assays and HPLC. Insome preferred embodiments, the variant has one or a limited number ofamino acid substitutions (e.g., conservative or non-conservativesubstitutions), additions, or deletions (e.g., truncations) compared towild type enzyme.

As used herein, the term “variant of a ribonuclease having similarimmunogenicity properties” refers to a variant of a ribonuclease (e.g.,SEQ ID NO:1) that, in some embodiments, exhibits substantially the sameor better immunogenicity properties than the non-modified ribonucleasefrom which it was derived. Immunogenicity properties include toxicityand undesirable immune responses (e.g., cytotoxic immune response) inanimals. In preferred embodiments, variants exhibit less than 100%,preferably less than 90%, even more preferably less than 80%, and stillmore preferably less than 70% of the toxicity or undesirable immuneresponse of the non-modified ribonuclease from which it was derived. Thelevel of toxicity or immunogenicity can be determined using any suitablemethod including, but not limited to, commercially available assays fortoxicity and immune response (e.g., measurement of cytokines or T-cellresponse). In some preferred embodiments, the variant has one or alimited number of amino acid substitutions (e.g., conservative ornon-conservative substitutions), additions, or deletions (e.g.,truncations) compared to wild type enzyme.

The term “heterologous nucleic acid sequence” or “heterologous gene” areused interchangeably to refer to a nucleotide sequence which is ligatedto a nucleic acid sequence to which it is not ligated in nature, or towhich it is ligated at a different location in nature. Heterologous DNAis not endogenous to the cell into which it is introduced, but has beenobtained from another cell. Generally, although not necessarily, suchheterologous DNA encodes RNA and proteins that are not normally producedby the cell into which it is expressed. Examples of heterologous DNAinclude reporter genes, transcriptional and translational regulatorysequences, selectable marker proteins (e.g., proteins which confer drugresistance or therapeutic benefits), etc.

As used herein, the term “immunoglobulin” or “antibody” refer toproteins that bind a specific antigen. Immunoglobulins include, but arenot limited to, polyclonal, monoclonal, chimeric, and humanizedantibodies, Fab fragments, F(ab′)₂ fragments, and includesimmunoglobulins of the following classes: IgG, IgA, IgM, IgD, IgE, andsecreted immunoglobulins (sIg). Immunoglobulins generally comprise twoidentical heavy chains and two light chains. However, the terms“antibody” and “immunoglobulin” also encompass single chain antibodiesand two chain antibodies.

As used herein, the term “antigen binding protein” refers to proteinsthat bind to a specific antigen. “Antigen binding proteins” include, butare not limited to, immunoglobulins, including polyclonal, monoclonal,chimeric, and humanized antibodies; Fab fragments, F(ab′)₂ fragments,and Fab expression libraries; and single chain antibodies.

The term “epitope” as used herein refers to that portion of an antigenthat makes contact with a particular immunoglobulin.

When a protein or fragment of a protein is used to immunize a hostanimal, numerous regions of the protein may induce the production ofantibodies which bind specifically to a given region orthree-dimensional structure on the protein; these regions or structuresare referred to as “antigenic determinants”. An antigenic determinantmay compete with the intact antigen (i.e., the “immunogen” used toelicit the immune response) for binding to an antibody.

The terms “specific binding” or “specifically binding” when used inreference to the interaction of an antibody and a protein or peptidemeans that the interaction is dependent upon the presence of aparticular structure (i.e., the antigenic determinant or epitope) on theprotein; in other words the antibody is recognizing and binding to aspecific protein structure rather than to proteins in general. Forexample, if an antibody is specific for epitope “A,” the presence of aprotein containing epitope A (or free, unlabelled A) in a reactioncontaining labeled “A” and the antibody will reduce the amount oflabeled A bound to the antibody.

As used herein, the terms “non-specific binding” and “backgroundbinding” when used in reference to the interaction of an antibody and aprotein or peptide refer to an interaction that is not dependent on thepresence of a particular structure (i.e., the antibody is binding toproteins in general rather that a particular structure such as anepitope).

As used herein, the term “subject” refers to any animal (e.g., amammal), including, but not limited to, humans, non-human primates,rodents, and the like, which is to be the recipient of a particulartreatment. Typically, the terms “subject” and “patient” are usedinterchangeably herein in reference to a human subject.

As used herein, the term “subject suspected of having cancer” refers toa subject that presents one or more symptoms indicative of a cancer(e.g., a noticeable lump or mass) or is being screened for a cancer(e.g., during a routine physical). A subject suspected of having cancermay also have one or more risk factors. A subject suspected of havingcancer has generally not been tested for cancer. However, a “subjectsuspected of having cancer” encompasses an individual who has received apreliminary diagnosis (e.g., a CT scan showing a mass) but for whom aconfirmatory test (e.g., biopsy and/or histology) has not been done orfor whom the stage of cancer is not known. The term further includessubjects who once had cancer (e.g., an individual in remission). A“subject suspected of having cancer” is sometimes diagnosed with cancerand is sometimes found to not have cancer.

As used herein, the term “subject diagnosed with a cancer” refers to asubject who has been tested and found to have cancerous cells. Thecancer may be diagnosed using any suitable method, including but notlimited to, biopsy, x-ray, blood test, and the diagnostic methods of thepresent invention. A “preliminary diagnosis” is one based only on visual(e.g., CT scan or the presence of a lump) and antigen tests.

As used herein, the term “subject at risk for cancer” refers to asubject with one or more risk factors for developing a specific cancer.Risk factors include, but are not limited to, gender, age, geneticpredisposition, environmental exposure, and previous incidents ofcancer, preexisting non-cancer diseases, and lifestyle.

As used herein, the term “non-human animals” refers to all non-humananimals including, but are not limited to, vertebrates such as rodents,non-human primates, ovines, bovines, ruminants, lagomorphs, porcines,caprines, equines, canines, felines, ayes, etc.

“Amino acid sequence” and terms such as “polypeptide” or “protein” arenot meant to limit the amino acid sequence to the complete, native aminoacid sequence associated with the recited protein molecule.

The terms “test compound” and “candidate compound” refer to any chemicalor biological entity, pharmaceutical, drug, and the like that is acandidate for use to treat or prevent a disease, illness, sickness, ordisorder of bodily function (e.g., cancer). Test compounds comprise bothknown and potential therapeutic compounds. A test compound can bedetermined to be therapeutic by screening using the screening methods ofthe present invention.

As used herein, the term “sample” is used in its broadest sense. In onesense, it is meant to include a specimen or culture obtained from anysource, as well as biological and environmental samples. Biologicalsamples may be obtained from animals (including humans) and encompassfluids, solids, tissues, and gases. Biological samples include bloodproducts, such as plasma, serum and the like. Environmental samplesinclude environmental material such as surface matter, soil, water andindustrial samples. Such examples are not however to be construed aslimiting the sample types applicable to the present invention.

The term “gene” refers to a nucleic acid (e.g., DNA) sequence thatcomprises coding sequences necessary for the production of a polypeptideor precursor (e.g., ribonucleases or ribonuclease conjugates of thepresent invention). The polypeptide can be encoded by a full lengthcoding sequence or by any portion of the coding sequence so long as thedesired activity or functional properties (e.g., enzymatic activity,etc.) of the full-length or fragment are retained. The term alsoencompasses the coding region of a structural gene and the includingsequences located adjacent to the coding region on both the 5′ and 3′ends for a distance of about 1 kb on either end such that the genecorresponds to the length of the full-length mRNA. The sequences thatare located 5′ of the coding region and which are present on the mRNAare referred to as 5′ untranslated sequences. The sequences that arelocated 3′ or downstream of the coding region and that are present onthe mRNA are referred to as 3′ untranslated sequences. The term “gene”encompasses both cDNA and genomic forms of a gene. A genomic form orclone of a gene contains the coding region interrupted with non-codingsequences termed “introns” or “intervening regions” or “interveningsequences.” Introns are segments of a gene that are transcribed intonuclear RNA (hnRNA); introns may contain regulatory elements such asenhancers. Introns are removed or “spliced out” from the nuclear orprimary transcript; introns therefore are absent in the messenger RNA(mRNA) transcript. The mRNA functions during translation to specify thesequence or order of amino acids in a nascent polypeptide.

The term “fragment” as used herein refers to a polypeptide that has anamino-terminal and/or carboxy-terminal deletion as compared to thenative protein, but where the remaining amino acid sequence is identicalto the corresponding positions in the amino acid sequence deduced from afull-length cDNA sequence. Fragments typically are at least 4 aminoacids long, preferably at least 20 amino acids long, usually at least 50amino acids long or longer, and span the portion of the polypeptiderequired for intermolecular binding of the compositions with its variousligands and/or substrates. In some embodiments, fragments posses anactivity of the native protein.

As used herein, the term “purified” or “to purify” refers to the removalof impurities and contaminants from a sample. For example, antibodiesare purified by removal of non-immunoglobulin proteins; they are alsopurified by the removal of immunoglobulin that does not bind an intendedtarget molecule. The removal of non-immunoglobulin proteins and/or theremoval of immunoglobulins that do not bind an intended target moleculeresults in an increase in the percent of target-reactive immunoglobulinsin the sample. In another example, recombinant polypeptides areexpressed in host cells and the polypeptides are purified by the removalof host cell proteins; the percent of recombinant polypeptides isthereby increased in the sample.

The term “expression vector” as used herein refers to a recombinant DNAmolecule containing a desired coding sequence and appropriate nucleicacid sequences necessary for the expression of the operably linkedcoding sequence in a particular host organism. Nucleic acid sequencesnecessary for expression in prokaryotes usually include a promoter, anoperator (optional), and a ribosome-binding site, often along with othersequences. Eukaryotic cells are known to utilize promoters, enhancers,and termination and polyadenylation signals.

As used herein, the term “host cell” refers to any eukaryotic orprokaryotic cell (e.g., bacterial cells such as E. coli, yeast cells,mammalian cells, avian cells, amphibian cells, plant cells, fish cells,and insect cells), whether located in vitro or in vivo. For example,host cells may be located in a transgenic animal.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides ribonuclease compositions and methods.The ribonucleases find use as pharmaceutical agents and as researchagents (e.g., for studying or characterizing biological processes incells, tissues, or organisms). For example, in some embodiments,ribonucleases comprising SEQ ID NO:1 are provided. SEQ ID NO:1 is thenative human RNase 1 with amino acid modifications: R4C, G38R, R39G,N67R, G89R, S90R, and V118C (X#Y, where X is the native amino acid, # isthe amino acid position based on a recognized numbering system for humanRNase 1, and Y is the modified amino acid that replaces X). The presentinvention also provides variants of SEQ ID NO:1 that maintain the 4C,38R, 39G, 67R, 90R, and 118C sequences, but include one or moreadditional amino acid changes. A variety of variants are described inmore detail below.

The ribonucleases of the invention were identified based on a balance ofa number of properties to yield an effective therapeutic molecule.Properties considered included stability, cytotoxicity towardspathogenic cells, efficacy of degradation of pathogenic RNA of anyorigin including viral RNA, evasion of binding by RNase inhibitors,resistance to degradation by proteases, delivery to target cells,efficiency of import into the cell, dose response properties,pharmacokinetic properties, and longevity within the human body. Forexample, the ribonuclease of SEQ ID NO:1 provides a useful therapeuticmolecule that has a balance of properties making it superior to priordescribed therapeutic ribonucleases, such as QBI-119 described in U.S.Pat. Publ. Ser. No. 20050261232 (herein incorporated by reference in itsentirety). The ribonuclease of SEQ ID NO:1 is inferior to QBI-119 andnative human RNase 1 in certain characteristics that might be consideredimportant in selecting an optimal compound (e.g., enzyme activity,ribonuclease inhibitor evasion, etc.), yet provides an overall superiortherapeutic agent. For example, SEQ ID NO:1 has only a fraction of theof the ribonuclease activity of native human RNase1 (less than 1%), yetis therapeutically effective.

The ribonucleases of the present invention find use in treating a widevariety of disease states, including a wide variety of cancer types. Forexample, SEQ ID NO:1 was tested against non-small cell lung cancer cells(A549 cell line), pancreatic cancer cells (BxPC3 cell line), andprostate cancer cells (DU145 cell line) and found to inhibit tumorgrowth greater than 65% (and as high as 94%) with dose ranges of 15mg/kg-100 mg/kg given 1 to 5 times per day or week. This was superior toQBI-119 both with respect to the percent of inhibition (which was equalto or better than QBI-119) and the diversity of cancer cell types atwhich it was highly efficacious.

The ribonucleases of the present invention may be formulated in anydesired form with or without other therapeutic agents, carriers,excipients, or other components (e.g., targeting moieties, water-solublemolecules, and the like). The ribonucleases may be packaged into kits,containing packaging and appropriate containers for shipment and/orstorage as therapeutic or research agents, including preparation intodosage form for single or multiple doses for one or more patients.

The present invention further provides variants of SEQ ID NO:1,including amino acid variations that do not significantly alter one ormore or any therapeutically relevant properties of the ribonuclease.Examples of these variants are set forth in more detail herein. Forexample, it is contemplated that isolated replacement of a leucine withan isoleucine or valine, an aspartate with a glutamate, a threonine witha serine, or a similar replacement of an amino acid with a structurallyrelated amino acid (i.e., conservative mutations) will not have a majoreffect on the biological activity of the resulting molecule.Accordingly, some embodiments of the present invention provide variantsof ribonucleases disclosed herein containing conservative replacements.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Genetically encodedamino acids can be divided into four families: (1) acidic (aspartate,glutamate); (2) basic (lysine, arginine, histidine); (3) nonpolar(alanine, valine, leucine, isoleucine, proline, phenylalanine,methionine, tryptophan); and (4) uncharged polar (glycine, asparagine,glutamine, cysteine, serine, threonine, tyrosine). Phenylalanine,tryptophan, and tyrosine are sometimes classified jointly as aromaticamino acids. In similar fashion, the amino acid repertoire can begrouped as (1) acidic (aspartate, glutamate); (2) basic (lysine,arginine, histidine), (3) aliphatic (glycine, alanine, valine, leucine,isoleucine, serine, threonine), with serine and threonine optionally begrouped separately as aliphatic-hydroxyl; (4) aromatic (phenylalanine,tyrosine, tryptophan); (5) amide (asparagine, glutamine); and (6)sulfur-containing (cysteine and methionine) (e.g., Stryer ed.,Biochemistry, pg. 17-21, 2nd ed, WH Freeman and Co., 1981). Whether achange in the amino acid sequence of a peptide results in a functionalpolypeptide can be readily determined by assessing the ability of thevariant peptide to function in a fashion similar to the wild-typeprotein. Peptides having more than one replacement can readily be testedin the same manner.

More rarely, a variant includes “nonconservative” changes (e.g.,replacement of a glycine with a tryptophan). Analogous minor variationscan also include amino acid deletions or insertions, or both. Guidancein determining which amino acid residues can be substituted, inserted,or deleted without abolishing biological activity can be found usingcomputer programs (including, but not limited to, FADE (Mitchell et al.,(2004). Molec. Simul. 30, 97-106); MAPS (Ban et al., Proceedings of the8th Annual International Conference on Research in ComputationalMolecular Biology, 2004, 205-212), SYBYL (Tripos, Inc, St. Louis, Mo.);and PyMOL (available on the Internet web site of sourceforge)).

Crystal structures of RNase 1 are described, for example in Pous et al.(Acta Crystallogr D Biol Crystallogr. 2001; 57, 498-505) and Pous et al.(J Mol. Biol. 2000; 303, 49-60) and serve as the basis for selection ofchanges. In addition, crystal structures are available for other humanpancreatic ribonucleases, including eosinophil derived neurotoxin (EDN,RNase 2; Swaminathan et al, Biochemistry, 2002, 41, 3341-3352, Mosimannet al J. Mol. Biol., 1996, 260, 540-552.; Iyer et al J Mol Biol, 2005,347, 637-655), eosinophil cationic protein (ECP, RNase 3; Mohan et alBiochemistry 2002; 41, 12100-12106; Boix et al Biochemistry, 1999, 38,16794-16801.; Mallorqui-Fernandez et al J. Mol. Biol, 2000, 300,1297-1307), RNase 4 (Terzyan et al, 1999, 285, 205-214.) and angiogenin(RNase 5; Leonidas et al J. Mol. Biol. 1999, 285, 1209-1233; Leonidas etal Protein Sci., 2001, 10, 1669-1676; Papageorgiou et al EMBO J., 1997,16, 5162-5177; Shapiro et al J. Mol. Biol., 2000, 302, 497-519.).

Variants may be produced by methods such as directed evolution or othertechniques for producing combinatorial libraries of variants, describedin more detail below. In still other embodiments of the presentinvention, the nucleotide sequences of the present invention may beengineered in order to alter a human RNase coding sequence including,but not limited to, alterations that modify the cloning, processing,localization, secretion, and/or expression of the gene product. Forexample, mutations may be introduced using techniques that are wellknown in the art (e.g., site-directed mutagenesis to insert newrestriction sites, alter glycosylation patterns, or change codonpreference, etc.). In some embodiments, changes are made in the nucleicacid sequence encoding a polypeptide of the present invention in orderto optimize codon usage to the organism that the gene is expressed in.

Exemplary variants are described below, including, but not limited to,substitutions, truncations, chimeras, etc. The present invention is notlimited to these particular variants. Both variants in the active siteand substrate-binding region and away from the active site arecontemplated to be within the scope of the present invention. Forexample, variants of SEQ ID NO:1 include the R4C, G38R, R39G, N67R,G89R, S90R, and V118C changes compared to normal human RNAse 1, but alsocontain one or more additional changes that either impact or do notimpact one or more of the activities or properties of the enzyme.Variants may be selected based on, for example, experimental data,computer modeling, and by rational design by comparison to otherribonucleases. Activities may be tested using assays to select thevariants with the desired properties (see e.g., Raines et al., J. Biol.Chem., 273, 34134 (1998); Fisher et al., Biochemistry 37:12121 (1998);Guar et al., J. Biol. Chem., 276:24978 (2001); Bosch, et al.,Biochemistry, 43:2167 (2004,); Lin, J. Biol. Chem., 245:6726 (1970); Balet al, Eur. J. Biochem., 245:465 (1997); Guar et al., Mol. Cell.Biochem., 275:95 (2005); Benito et al., Protein Eng., 15:887 (2002);Ribo et al., Biol. Chem. Hoppe-seyler, 375:357 (1994); DiGaetano et al.,Biochem. J., 358:241 (2001); Trautwein et al., FEBS Lett., 281:277(1991); Curran et al., Biochemistry 32:2307 (1993); Sorrentino et al.,Biochemistry 42:10182 (2003); herein incorporated by reference in theirentireties).

Exemplary amino acid locations for modification in the production ofvariants are provided below. One or more sites may be modified, asdesired. The exemplary list below provides a ranking of the utility formodification of each amino acid position based on the amino acidnumbering of a wild-type human RNase 1 (e.g., as represented by interestin modifying (e.g., so as to result in a functional ribonuclease (e.g.,comprising a desired property (e.g., cancer cell killing and/orribonucleolytic activity))). An “interest site” may be characterized asa “high interest site,” a “medium interest site,” or a “low interestsite” based on characteristics of the ribonuclease described herein(e.g., biologic activity (e.g., ribonucleolytic activity, cancer cellkilling activity, oligomerization capacity, etc.)) desired to beretained within the ribonuclease after modification of the same (e.g.,for deletion, substitution or other type of mutation to create aribonuclease variant, and/or for conjugation to a water-solublepolymer). For example, high interest sites are those that can bemodified without significantly interfering with one or more desiredactivities or properties of the ribonuclease: High interest (1-3, 13-24,31-39, 48-50, 60, 66-71, 75-78, 87-94, 105, 112-115, and 127-128);Medium interest (4-11, 25, 27-30, 42-47, 51-57, 59, 61-64, 73-74, 79-83,85-86, 96-104, 106-109, 111, 116-118, and 120-126); and Low interest(12, 26, 40-41, 58, 65, 72, 84, 95, 110, and 119).

It will be appreciated that one or more modification sites may be used.Preferably, the selected sites are high or medium interest sites.However, one or more additional sites may be altered as desired andappropriate for the intended application. It should be noted that, insome embodiments, RNase is produced in such a way that a methionine(e.g., that is not part of wild type human RNase) is incorporated as thefirst amino acid of the protein (e.g., via the methods used to producethe protein (e.g., recombinant human ribonuclease (e.g., produced in E.coli))). Thus, in some embodiments, the numbering of amino acid residuesdepicted herein may be off by a numerical value of one (e.g., if amethionine is incorporated into the protein, then the numbering of theamino acid residues of the human RNases recited herein is off by 1(i.e., because a methionine is incorporated in position 1, the numberingof the amino acids depicted will be short by one, e.g., the residuenumber 10 would actually be residue number 11 because of the methionineincorporated at position 1)). Similarly, the positions depicted may alsobe applied to corresponding numerical positions other relatedribonucleases.

In some embodiments, the desired residues for modification (e.g.,deletion, mutation, etc.) in the present invention are selected to avoiddisruption of the tertiary structure and/or stability of theribonuclease. In some embodiments, these residues are on the surface ofthe protein (e.g., residues generally exposed to solvent (e.g., water orbuffer)). For example, in some embodiments, the types of residues thatare modified include, but are not limited to, amino acids that appeardisordered in crystal structures, residues that contact the ribonucleaseinhibitor protein, and amino acids not involved in tertiary structures(e.g., alpha helices and beta sheets), amino acids in loop regionsbetween structures (e.g. alpha helices and beta sheets) as well as aminoacids towards the end of the protein (the N- and C-termini). In someembodiments, additional amino acid residues are added to either the N-or C-terminus (e.g., to generate a RNase analogue and/or for conjugationof a water-soluble polymer).

In some embodiments, the present invention provides polymer conjugationof ribonucleases to increase its circulating half-life in vivo whileretaining ribonucleolytic activity or other desired function (e.g.,cancer cell killing). In some embodiments, the ribonuclease isconjugated to a water-soluble polymer in a region of the proteininvolved in evasion from ribonuclease inhibitor (RI). In some preferredembodiments, the ribonuclease is conjugated to a water-soluble polymerin a region of the protein that is not involved in evasion from RI(e.g., a region that has no impact on binding of the ribonuclease to theRI). Examples of regions that are not involved in evasion from RIinclude, but are not limited to, regions comprising amino acid residuesat positions 1, 49, 75 or 113. Thus, although an understanding of themechanism is not necessary to practice the present invention and thepresent invention is not limited to any particular mechanism of action,in some embodiments, conjugation of a water soluble polymer toribonuclease possessing biological activity (e.g., cancer cell killing)even though the conjugation does not assist the ribonuclease fromevading the RI.

In some embodiments, the present invention utilizes incorporation of aunique functional group in RNases for conjugation of a water-solublepolymer. For example, in some embodiments, a cysteine molecule isengineered into a RNase (e.g., without loss of ribonucleolytic activityor other desired function (e.g., cancer cell killing capacity)) in orderto provide a free thiol group for conjugation to a water-solublepolymer. Free thiol groups are not found elsewhere in the RNase therebyproviding the ability to generate a homogenous conjugation. In otherembodiments, recombinant DNA technology is utilized to provide modifiedor novel codons to incorporate non-natural amino acids with orthogonalfunctionality into the RNase of interest (e.g., without loss ofribonucleolytic activity).

In some embodiments, ribonucleases are modified to include one or moreamino acid residues such as, for example, lysine, cysteine and/orarginine, in order to provide an attachment location for water-solublepolymer (e.g., to an atom within the side chain of the amino acid).Techniques for adding amino acid residues are well known to those ofordinary skill in the art (See, e.g., March, Advanced Organic Chemistry:Reactions Mechanisms and Structure, 4th Ed. (New York:Wiley-Interscience, 1992).

The present invention is not limited by the type of modification made tosaid ribonuclease described herein. In some embodiments, the presentinvention is modified through the attachment of one or more moietiesselected from the group comprising dextran, carbohydrate, albumin,carrier protein, and antibody (e.g., a non-targeting antibody used toextend the half-life of the ribonuclease).

The present invention is not limited by the type of water-solublepolymer utilized for conjugation to a human ribonuclease describedherein. Indeed, any biocompatible water-soluble polymer may be used. Insome embodiments, the water-soluble polymer is nonpeptidic, nontoxic,non-naturally occurring and/or biocompatible. A water-soluble polymer isconsidered biocompatible if the beneficial effects associated with useof the polymer alone or with another substance (e.g., conjugated to theribonuclease) in connection with living tissues (e.g., administration toa patient) outweighs any deleterious effects as evaluated by a clinician(e.g., a physician). With respect to non-immunogenicity, a polymer isconsidered nonimmunogenic if the intended use of the polymer in vivodoes not produce an undesired immune response (e.g., the formation ofantibodies) or, if an immune response is produced, that such a responseis not deemed clinically significant or important as evaluated by aclinician. Thus, in some preferred embodiments, the water-solublepolymer is biocompatible and nonimmunogenic.

Water-soluble polymers of the present invention are selected such that,when attached to a human ribonuclease, the polymer does not precipitatein an aqueous environment, such as a physiological environment. In someembodiments, the polymer is selected based upon the method ofconjugation to the human ribonuclease protein. For example, for methodsutilizing reductive alkylation, the polymer selected should have asingle reactive aldehyde so that the degree of polymerization may becontrolled. The polymer may be branched or unbranched. Preferably, fortherapeutic use of the end-product preparation, the polymer will bepharmaceutically acceptable. One skilled in the art will be able toselect the desired polymer based on such considerations as whether thepolymer/protein conjugate will be used therapeutically, and if so, thedesired dosage, circulation time, resistance to proteolysis, and otherconsiderations. For example, these may be ascertained by assaying forribonucleolytic activity of the conjugate in vitro using methods wellknown in the art.

The water-soluble polymer may be selected from the group including, butnot limited to, poly(alkylene glycols) such as polyethylene glycol(PEG), poly(propylene glycol) (“PPG”), copolymers of ethylene glycol andpropylene glycol and the like, poly(oxyethylated polyol), poly(olefinicalcohol), poly(vinylpyrrolidone), poly(hydroxyalkylmethacrylamide),poly(hydroxyalkylmethacrylate), poly(saccharides), poly(.alpha.-hydroxyacid), poly(vinyl alcohol), polyphosphazene, polyoxazoline,poly(N-acryloylmorpholine), and combinations of any of the foregoing.

The polymer may be linear (e.g., alkoxy PEG or bifunctional PEG) orbranched. Furthermore, the polymer may be multi-armed (e.g., forked PEGor PEG attached to a polyol core), dendritic, and/or comprise degradablelinkages. It is contemplated that the internal structure of the polymercan be organized in any of a number of different patterns (e.g.,patterns including, but not limited to, homopolymer, alternatingcopolymer, random copolymer, block copolymer, alternating tripolymer,random tripolymer, and block tripolymer).

Furthermore, the polymer may be “activated” with a suitable activatinggroup appropriate for coupling to a desired residue within theribonuclease. An “activated” polymer refers to a polymer that possessesreactive groups for reaction with a ribonuclease. Examples of activatedpolymers and methods for their conjugation to proteins that arecontemplated to be useful (e.g., for conjugating a water-soluble polymerto a human ribonuclease) in the present invention are known in the artand are described in detail in Zalipsky, Bioconjugate Chem 6, 150-165(1995); Kinstler et al., Advanced Drug Delivery Reviews 54, 477-485(2002); and Roberts et al., Advanced Drug Delivery Reviews 54, 459-476(2002); each of which is hereby incorporated by reference in itsentirety for all purposes.

The polymer may be of any molecular weight. For example, forpolyethylene glycol, a preferred molecular weight is between about 2 kDaand about 150 kDa (the term “about” indicating that in preparations ofpolyethylene glycol, some molecules will weigh more, some less, than thestated molecular weight). Other sizes may be used, depending on thedesired therapeutic profile (e.g., the duration of sustained releasedesired, the effects, if any on biological activity, the ease inhandling, the degree or lack of antigenicity and other known effects ofthe polyethylene glycol to a therapeutic composition of the presentinvention (e.g., comprising a RNase protein or analog)).

When polyethylene glycol (PEG) is utilized as the water soluble polymer,PEG may have one of its termini capped with an inert group. For example,the PEG molecule may be methoxy-PEG, also referred to as mPEG, which isa form of PEG wherein one terminus of the polymer is a methoxy (e.g.,—OCH₃) group, while the other terminus is a functional group (e.g.,hydroxyl) that can be chemically modified and used for conjugation to areactive group on a target protein (e.g., human ribonuclease). In someembodiments, a PEG polymer described in U.S. Pat. App. Pub. No.20040235734 is used, herein incorporated by reference in its entirety.

In some embodiments, the PEG polymer may comprise one or more weak ordegradable linkages. For example, a PEG polymer may comprise an esterlinkage (e.g., that may hydrolyze over time (e.g., when present within apatient)). In some embodiments, hydrolysis of the PEG polymer comprisinga degradable linkage produces two or more fragments (e.g., of lowermolecular weight than the parent molecule).

The present invention is not limited by the type of degradable linkage.Indeed, a PEG polymer may comprise one or more of a variety ofdegradable linkages including, but not limited to, carbonate linkages;imine linkages; phosphate ester linkages; hydrazone linkages; acetallinkages; orthoester linkages; amide linkages, urethane linkages;peptide linkages; and oligonucleotide linkages.

It is contemplated that the inclusion of one or more degradable linkageswithin the polymer itself provides an added mechanism to control thepharmacokinetic characteristics of the conjugates of the presentinvention. For example, in some embodiments, a RNase-PEG conjugate ofthe present invention may be administered to a patient wherein theconjugate, when administered, possesses little to no enzymatic activity,but when exposed to conditions such that the linkages degrade (e.g.,hydrolyze), the ribonucleolytic activity of the enzyme is activated.Thus, in some embodiments, the degradable linkages within the PEGmolecule can be used for increasing specificity and efficacy of theconjugate.

It is contemplated that the conjugates of the present invention maycomprise a linkage between the polymer (e.g., PEG) and humanribonuclease protein. In some embodiments, the linkage is a stablelinkage (e.g., amide linkage, carbamate linkage, amine linkage,thioether/sulfide linkage, or carbamide linkage. In some embodiments,the linkage is hydrolytically degradable (e.g., to allow release of theRNase (e.g., without a portion of the polymer (e.g., PEG) remaining onthe RNase)). The present invention is not limited by the type ofdegradable linkage utilized. Indeed, a variety of linkages arecontemplated herein including, but not limited to, carboxylate ester,phosphate ester, thiolester, anhydrides, acetals, ketals, acyloxyalkylether, imines, orthoesters, peptides and oligonucleotides. Theselinkages may be prepared by modification of either the RNase protein(e.g., at the C-terminal carboxyl group, or a hydroxyl group of an aminoacid side chain) and/or the polymer (e.g., using methods known in theart).

The proportion of water-soluble polymer (e.g., PEG) to ribonucleaseprotein molecules may vary, as may their concentrations in the reactionmixture. In general, the optimum ratio (e.g., in terms of efficiency ofreaction (e.g., to conjugate polymer to one, two three, four or moresites) where there is little to no excess unreacted protein or polymer)can be determined (e.g., using the molecular weight of the polymer(e.g., PEG) selected, conjugation chemistry utilized, number of interestsites targeted, etc.). For example, in some embodiments, a non-specificconjugation reaction (e.g., PEGylation reaction) can be carried outfollowed by a later purification (e.g., to separate RNases based uponthe number of polymers (e.g., PEGs) conjugated to each RNase).

In some embodiments, the conjugates are present within a composition.For example, in some embodiments, the composition comprises a pluralityof conjugates, wherein each protein comprises 1-3 water-soluble polymerscovalently attached to the protein. In some embodiments, the compositioncomprises a plurality of conjugates, wherein each protein comprise 1, 2,3, 4, 5, 6, or more polymers attached to the protein. In someembodiments, the composition comprises a population of conjugateswherein the majority of conjugates (e.g., greater than 65%, greater than70%, greater than 75%, greater than 80%, greater than 85%, greater than90%, greater than 95%, greater than 97%, greater than 98%, greater than99%) are covalently attached to the same number (e.g., 1, 2, 3, or more)of polymers (e.g., PEG molecules). In some embodiments, 1, 2, 3, or morepolymers are conjugated to an oligomerized ribonuclease. The presentinvention is not limited by the number of ribonuclease molecules presentwithin an oligomer. Indeed, a variety of oligomers may be conjugated toone or more water-soluble polymers including, but not limited to,oligomers of two, three, four, five, six, or even more ribonucleases. Insome embodiments, the present invention provides a compositioncomprising a plurality of RNases that comprise a single water-solublepolymer (e.g., that are monoPEGylated). In some embodiments, theplurality of RNases comprise monomers, dimers, trimers, and/or higherorder complexes (i.e., oligomers) of RNases.

In preferred embodiments, the modified human ribonuclease proteins(e.g., water-soluble polymer-RNase conjugates) of the present inventionretain a significant portion of enzymatic (e.g., ribonucleolytic)activity. In some embodiments, the conjugate possesses from about 1% toabout 95% of the enzymatic activity of the unmodified (e.g.,non-conjugated) ribonuclease. In some embodiments, the conjugatepossesses more activity than the unmodified ribonuclease. In someembodiments, a modified human ribonuclease possesses about 1%, 5%, 10%,15%, 20%, 30%, 40%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%,97%, 99%, 100%, or more relative to that of the unmodified parentribonuclease possessing ribonucleolytic activity (e.g., as measured inan in vitro assay well known to those of skill in the art).

In other preferred embodiments, the modified human ribonuclease proteins(e.g., water-soluble polymer-RNase conjugates) of the present inventionretain a significant portion of another desired property (e.g., otherthan ribonucleolytic activity (e.g., cancer cell killing capacity)).Although an understanding of the mechanism is not necessary to practicethe present invention and the present invention is not limited to anyparticular mechanism of action, in some embodiments, a modified humanribonuclease protein (e.g., water-soluble polymer-RNase conjugate) iscapable of killing target cells (e.g., cancer cells or microbially(e.g., virally) infected cells) in the absence of (e.g., less than 70%,less than 60%, less than 50%, less than 40%, less than 30%, less than20%, less than 10%, or less than 5% of unmodified ribonuclease)ribonucleolytic activity (e.g., due to other characteristics of thehuman ribonuclease protein).

The present invention is not limited by the method utilized forconjugating a water-soluble polymer to a human ribonuclease of thepresent invention. Multiple types of chemistries are known in the artand may find use in the generation of the compositions of the presentinvention. These methods have been describe in detail (See, e.g.,Zalipsky, Bioconjugate Chem 6, 150-165 (1995); Kinstler et al., AdvancedDrug Delivery Reviews 54, 477-485 (2002); and Roberts et al., AdvancedDrug Delivery Reviews 54, 459-476 (2002)). In some embodiments, thepresent invention utilizes a conjugation chemistry useful forconjugating an activated polymer of the present invention to a humanribonuclease.

For example, for obtaining N-terminally conjugated RNase (e.g.,N-terminally PEGylated RNase), reductive alkylation may be used. Amethod for attaching without a linking group between the polymer (e.g.,PEG) and the protein moiety is described in Francis et al., In:Stability of protein pharmaceuticals: in vivo pathways of degradationand strategies for protein stabilization (Eds. Ahern., T. and Manning,M. C.) Plenum, N.Y., 1991). In some embodiments, a method involving theuse of N-hydroxy succinimidyl esters of carboxymethyl mPEG is used (See,e.g., U.S. Pat. No. 5,824,784, issued Oct. 20, 1998, hereby incorporatedby reference in its entirety).

In some embodiments, the PEG-ribonuclease conjugate is purified afterconjugation. The present invention is not limited by the type ofpurification process utilized. Indeed, a variety of processes may beutilized including, but not limited to, gel filtration chromatography,ion exchange chromatography, hydrophobic interaction chromatography,size exclusion chromatography, and other methods well known in the art.

For example, in some embodiments, a water-soluble polymer-RNaseconjugate can be purified to obtain one or more different types ofconjugates (e.g., a conjugate covalently bound to a single polymer). Insome embodiments, the products of a conjugation reaction are purified toobtain (e.g., on average) anywhere from 1, 2, 3, 4, or more polymers(e.g., PEGs) per human ribonuclease. In some embodiments, gel filtrationchromatography is used to separate/fractionate ribonucleases covalentlyattached to different numbers of polymers or to separate a conjugatefrom non-conjugated protein or from non-conjugated polymer. Gelfiltration columns are well known in the art and available from multiplesources (e.g., SUPERDEX and SEPHADEX columns from Amersham Biosciences,Piscataway, N.J.).

In some embodiments, the present invention provides a compositioncomprising a water-soluble polymer-human ribonuclease conjugate. In someembodiments, the composition is administered to a patient in order totreat cancer. Thus, in some embodiments, the present invention providesa method of treating cancer comprising administering a compositioncomprising a water-soluble polymer-human ribonuclease conjugate.

In some embodiments, a therapeutic composition of the inventioncomprises an antibody and a ribonuclease. The antibody may provide cellor tissue specific targeting, and/or additional therapeutic benefits.The following table lists the mechanisms of some cancer therapeuticantibodies, including three antibody conjugates that carry a toxicpayload for lymphomas and leukemias. (Drug Discovery Today, Vol. 8, No.11 Jun. 2003). Two of the conjugates, ZEVALIN and BEXXAR, carryradioactive iodine as the toxin and the third, MYLOTARG, carries acytotoxic antitumor antibiotic, calicheaminin which is isolated from abacterial fermentation. The Mylotarg antibody binds specifically to theCD33 antigen which is expressed on the surface of leukemic blasts thatare found in more than 80% of patients with acute myeloid leukemia(AML). The antibody in this conjugate has approximately 98.3% of itsamino acid sequences derived from human origins.

TABLE 1 Antibody Mode of Action Product Antibody Target Blockade Ligandbinding ERBITUX EGF receptor HUMAX-EGFR EGF receptor ComplementDependent Cytotoxicity RITUXAN CD20 HUMAX-CD20 CD20 CAMPATH-1H CD52Antibody dependent cell-mediated RIXTUXAN CD20 cytotoxicity HUMAX-CD20CD20 HERCEPTIN Her-2/neu HUMAX-EGFR EGF receptor Apoptosis inductionVarious IdiotypeB cell tumors Disruption signaling 2C4 Her-2/neu(PERTUZUMAB) Inhibition angiogenesis AVASTIN VEGF Targeted radiolysisconjugate ZEVALIN CD20 BEXXAR CD20 Toxin-mediated killing by conjugateMYLOTARG CD33 Antagonist activity MDX-010 CTLA4 Agonist activity VariousCD40, CD137 Antagonist activity Preclinical MAb Epithelial cell receptorprotein tyrosine kinase (EphA2) Antagonist activity Phase II Mab alpha 5beta 3 integrin (receptor) Antagonist activity Phase I bispecificCD19/CD3 single chain monoclonal antibody Antagonist activityPreclinical MAb Interleukin 9 Antagonist activity RespiGam Respiratorysyncytial virus Polyclonal Antibody Antagonist activity Phase II MAb CD2Catalytic Activity Mab Cocaine cleavage Anti-infective, bacteria Mabbacteria Immunosuppressive Agents Mab Graft versus Host DiseaseAnti-infective, virus Mab Human metapneumovirus Cytostatic agent MabPlatelet derived growth factor Cancer growth and metastosis PreclinicalMAb Human beta hydroxylases Treatment of autoimmune disease MAb Medi 507Mixed lymphocyte responses Anti-infective, virus Polyclonal antibodycytomegalovirus Anti-idiotype antibody Mab Neu-glycolyl-GM3 gangliosideProdrug carrier Mab Immungen's CC 1065 prodrugs Toxin-mediated killingby conjugate Preclinical MAb Various by Immunogen and taxane derivativesToxin-mediated killing by conjugate Cantuzumab Can Ag receptor bymertansine immunogen conjugate Toxin-mediated killing by conjugate PhaseII MAb CD56 maytansinoid conjugate Toxin for mitosis inhibition MAbmaitansine various conjugate Toxin-mediated killing by conjugatePreclinical MAb Antigen on squamous cell cytotoxic drug cancer(Immunogen) DM1 conjugate

Any of the targeting antibodies or agents used in these products mayalso be employed by the compositions and methods of the presentinvention.

Generally, the most specific method for targeting toxins is the use ofmonoclonal antibodies or antibody fragments that are designed torecognize surface antigens specific to tumor cells. Because normal cellslack the surface antigens, they are not targeted and killed by the toxinconjugate. Whole antibodies have two domains: a variable domain thatgives the antibody its affinity and binding specificity and a constantdomain that interacts with other portions of the immune system tostimulate immune responses in the host organism. The variable domain iscomposed of the complementarity determining regions (CDRs), which bindto the antibody's target, and a framework region that anchors the CDRsto the rest of the antibody and helps maintain CDR shape. The six CDR'sin each antibody differ in length and sequence between differentantibodies and are mainly responsible for the specificity (recognition)and affinity (binding) of the antibodies to their target markers.

In addition to antibody delivery vectors, toxic molecules can bedelivered to cancer cells using several other specific and non-specificvectors including peptides, polymers, dendrimers, liposomes, polymericnanoparticles, and block copolymer micelles. For example, peptides thatbind to the leutinizing hormone-releasing hormone have been used totarget a small molecule toxin, camptothecin, to ovarian cancer cells(Journal of Controlled Release, 2003, 91, 61-73.).

Ribonucleases are effective toxins in human cells, particularly againstcancer cells. The following references, each of which is hereinincorporated by reference in its entirety, describe some chemicalconjugates of ribonucleases to targeting proteins (including proteinsand antibodies): Newton et al. (2001), Blood 97(2): 528-35, Hursey etal. (2002) Leuk Lymphoma 43(5): 953-9, Rybak et al., (1991) Journal ofBiological Chemistry 266(31): 21202-7, Newton et al. (1992) Journal ofBiological Chemistry 267(27): 19572-8, Jinno and Ueda (1996) CancerChemother Pharmacol 38: 303-308, Yamamura et al. (2002) Eur J Surg 168:49-54, Jinno et al. (1996) Life Sci 58: 1901-1908, Suzuki et al. (1999)Nature Biotechnology 17(3): 265-70, Rybak et al. (1992), Cell Biophys21(1-3): 121-38, Jinno et al. (2002) Anticancer Res. 22: 4141-4146.

In some embodiments, the present invention provides recombinantconstructs comprising a nucleic acid sequence that encodes one or morethe amino acid sequences described herein. In some embodiments of thepresent invention, the constructs comprise a vector, such as a plasmidor viral vector, into which a sequence of SEQ ID:NO 2, or relatedsequence, has been inserted, in a forward or reverse orientation. Instill other embodiments, the heterologous structural sequence isassembled in appropriate phase with translation initiation andtermination sequences.

In some embodiments of the present invention, the appropriate DNAsequence is inserted into the vector using any of a variety ofprocedures. In general, the DNA sequence is inserted into an appropriaterestriction endonuclease site(s) by procedures known in the art. Largenumbers of suitable vectors are known to those of skill in the art, andare commercially available. Such vectors include, but are not limitedto, the following vectors: 1) Bacterial—pQE70, pQE60, pQE-9 (Qiagen),pET 22b, pET26b, pET 30b (Novagen brand, EMD Chemicals), pBS, pD10,phagescript, psiX174, pbluescript SK, pBSKS, pNH8A, pNH16a, pNH18A,pNH46A (Stratagene); ptrc99a, pKK223-3, pKK233-3, pDR540, pRIT5(Pharmacia); and 2) Eukaryotic—pWLNEO, pSV2CAT, pOG44, PXT1, pSG(Stratagene) pSVK3, pBPV, pMSG, pSVL (Pharmacia). Any other plasmid orvector may be used as long as they are replicable and viable in thehost. In some embodiments of the present invention, mammalian expressionvectors comprise an origin of replication, a suitable promoter andenhancer, and also any necessary ribosome binding sites, polyadenylationsites, splice donor and acceptor sites, transcriptional terminationsequences, and 5′ flanking non-transcribed sequences. In otherembodiments, DNA sequences derived from the SV40 splice, andpolyadenylation sites may be used to provide the requirednon-transcribed genetic elements.

In certain embodiments of the present invention, the DNA sequence in theexpression vector is operatively linked to an appropriate expressioncontrol sequence(s) (promoter) to direct mRNA synthesis. Promotersuseful in the present invention include, but are not limited to, the LTRor SV40 promoter, the E. coli lac or trp, the phage lambda PL and PR, T3and T7 promoters, and the cytomegalovirus (CMV) immediate early, herpessimplex virus (HSV) thymidine kinase, and mouse metallothionein-Ipromoters and other promoters known to control expression of gene inprokaryotic or eukaryotic cells or their viruses. In other embodimentsof the present invention, recombinant expression vectors include originsof replication and selectable markers permitting transformation of thehost cell (e.g., dihydrofolate reductase or neomycin resistance foreukaryotic cell culture, or tetracycline, kanamycin, or ampicillinresistance in E. coli).

In some embodiments of the present invention, transcription of the DNAencoding the polypeptides of the present invention by higher eukaryotesis increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about from 10 to 300by that act on a promoter to increase its transcription. Enhancersuseful in the present invention include, but are not limited to, theSV40 enhancer on the late side of the replication origin by 100 to 270,a cytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenovirus enhancers.

In other embodiments, the expression vector also contains aribosome-binding site for translation initiation and a transcriptionterminator. In still other embodiments of the present invention, thevector may also include appropriate sequences for amplifying expression.

In a further embodiment, the present invention provides host cellscontaining the above-described constructs. In some embodiments of thepresent invention, the host cell is a higher eukaryotic cell (e.g., amammalian or insect cell). In other embodiments of the presentinvention, the host cell is a lower eukaryotic cell (e.g., a yeastcell). In still other embodiments of the present invention, the hostcell can be a prokaryotic cell (e.g., a bacterial cell). Specificexamples of host cells include, but are not limited to, Escherichiacoli, Salmonella typhimurium, Bacillus subtilis, and various specieswithin the genera Pseudomonas, Streptomyces, and Staphylococcus, as wellas Saccharomycees cerivisiae, Schizosaccharomycees pombe, Drosophila S2cells, Spodoptera Sf9 cells, Chinese hamster ovary (CHO) cells, COS-7lines of monkey kidney fibroblasts, (Gluzman, Cell 23:175 [1981]), C127,3T3, 293, 293T, HeLa and BHK cell lines.

The constructs in host cells can be used in a conventional manner toproduce the gene product encoded by the recombinant sequence. In someembodiments, introduction of the construct into the host cell can beaccomplished by calcium phosphate transfection, DEAE-Dextran mediatedtransfection, transformation, or electroporation. Alternatively, in someembodiments of the present invention, the polypeptides of the inventioncan be synthetically produced by conventional peptide synthesizers.

Proteins can be expressed in mammalian cells, yeast, bacteria, or othercells under the control of appropriate promoters. Cell-free translationsystems can also be employed to produce such proteins using RNAs derivedfrom the DNA constructs of the present invention. Appropriate cloningand expression vectors for use with prokaryotic and eukaryotic hosts aredescribed by Sambrook, et al., Molecular Cloning: A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989).

In some embodiments of the present invention, following transformationof a suitable host strain and growth of the host strain to anappropriate cell density, the selected promoter is induced byappropriate means (e.g., temperature shift or chemical induction) andcells are cultured for an additional period. In other embodiments of thepresent invention, cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification. In still other embodiments of thepresent invention, microbial cells employed in expression of proteinscan be disrupted by any convenient method, including freeze-thawcycling, sonication, mechanical disruption, or use of cell lysingagents.

An example of a suitable recombinant product method involves use of aplasmid conferring drug resistance such as ampicillin (e.g., pET22b) orkanamycin (e.g., pET-26b, pET-30b) and containing the gene coding forthe ribonuclease protein. The plasmid harboring the gene is transformedinto an E. coli cell line. Cells that can be used include, but are notlimited to, E. coli B or K strain and the related mutant strains such asHB101, JM109, DHa5, BL-21A1 and BL21DE3. Expression of the ribonucleaseprotein can be induced during fermentation by addition of carbohydratessuch as arabinose or lactose or their analogues includingisopropyl-beta-D-thiogalactopyranoside (IPTG). The ribonuclease proteincan be produced as a soluble product or in inclusion bodies. For solubleproduction, the protein is isolated and purified as below using columnchromatography. For inclusion body production, the cells aremechanically, enzymatically or/and chemically lysed. The cell pelletcontaining the inclusion bodies is separated from the soluble materialby multiple methods, including diafiltration or centrifugation. Theinclusion bodies are solubilized with a denaturant such as urea orguanidine hydrochloride. Reducing agents, such as low molecular weightthiols (e.g., dithiothreitol) or tris(2-carboxyethyl)phosphine can beadded. Addition of acid causes precipitate to form that can be removedfrom the solution by filtration and/or centrifugation.

The protein is folded by changing to a buffer that may include a varietyof agents to assist in protein folding. Examples of such reagentsinclude: arginine, polyethylene glycol, sulfobetaines, detergents,organic salts and/or chaotropes. The buffer is changed again forcompatibility with the first column chromatography step and precipitatecan be removed by filtration or centrifugation. The column stepsinclude: anion exchange, cation exchange and hydrophobic interaction.Examples of anion exchange resins include but are not limited to: QSepharose XL, UNO Q-1, Poros 50 HQ, Toyopearl QAE 550c, Separon HemaBio1000Q, Q-Cellthru Bigbeads Plus, Q Sepharose HP, Toyopearl SuperQ 650s,Macro-Prep 25Q, TSK-Gel Q-5PW-HR, Poros QE/M, Q Sepharose FF, Q HyperD20, Q Zirconia, Source 30Q, Fractogel EMD TMAE 650s, and Express-Ion Q.Examples of cation exchange resins include but are not limited to: SPSepharose XL, Poros 50 HS, Toyopearl SP 550c, SP Sepharose BB, Source30S, TSKGel SP-5PW-HR20, Toyopearl SP 650c, Heparin Sepharose FF, SPSepharose FF, CM Sepharose FF, Heparin Toyopearl 650m, SP Toyopearl650m, CM Toyopearl 650m, Ceramic Heparin HyperD M, Ceramic S HyperD 20,and Ceramic CM HyperD F. Examples of hydrophobic interaction resinsinclude but are not limited to: TSK-gel Phenyl-5PW, TSK-gel Ether-5PW,TSK-gel Butyl-NPR, Toyopearl Butyl-650S, Toyopearl Ether-650S, ToyopearlPhenyl-650S, Toyopearl Butyl-650M, Toyopearl Ether-650M, ToyopearlPhenyl-650M, Butyl-Sepharose 4 FF, Octyl-Sepharose 4 FF, Octyl-SepharoseCL-4B, and Phenyl-Sepharose 6 FF (high or low substitution). The finalproduct is exchanged into a neutral buffer for long term storage.

In some embodiments, the compositions and methods of the presentinvention are used to treat diseased cells, tissues, organs, orpathological conditions and/or disease states in a subject organism(e.g., a mammalian subject including, but not limited to, humans andveterinary animals), or in in vitro and/or ex vivo cells, tissues, andorgans. In this regard, various diseases and pathologies are amenable totreatment or prophylaxis using the present methods and compositions. Anon-limiting exemplary list of these diseases and conditions includes,but is not limited to, breast cancer, prostate cancer, lymphoma, skincancer, pancreatic cancer, colon cancer, melanoma, malignant melanoma,ovarian cancer, brain cancer, primary brain carcinoma, head-neck cancer,glioma, glioblastoma, liver cancer, bladder cancer, non-small cell lungcancer, head or neck carcinoma, breast carcinoma, ovarian carcinoma,lung carcinoma, small-cell lung carcinoma, Wilms' tumor, cervicalcarcinoma, testicular carcinoma, bladder carcinoma, pancreaticcarcinoma, stomach carcinoma, colon carcinoma, prostatic carcinoma,genitourinary carcinoma, thyroid carcinoma, esophageal carcinoma,myeloma, multiple myeloma, adrenal carcinoma, renal cell carcinoma,endometrial carcinoma, adrenal cortex carcinoma, malignant pancreaticinsulinoma, malignant carcinoid carcinoma, choriocarcinoma, mycosisfungoides, malignant hypercalcemia, cervical hyperplasia, leukemia,acute lymphocytic leukemia, chronic lymphocytic leukemia, acutemyelogenous leukemia, chronic myelogenous leukemia, chronic granulocyticleukemia, acute granulocytic leukemia, hairy cell leukemia,neuroblastoma, rhabdomyosarcoma, Kaposi's sarcoma, polycythemia vera,essential thrombocytosis, Hodgkin's disease, non-Hodgkin's lymphoma,soft-tissue sarcoma, osteogenic sarcoma, primary macroglobulinemia, andretinoblastoma, and the like, T and B cell mediated autoimmune diseases;inflammatory diseases; infections; hyperproliferative diseases; AIDS;degenerative conditions, vascular diseases, and the like. In someembodiments, the cancer cells being treated are metastatic. In otherembodiments, the RNases of the present invention target cancer stemcells.

In subjects who are determined to be at risk of having cancer, thecompositions of the present invention are administered to the subjectpreferably under conditions effective to decrease angiogenesis,proliferation and/or induce killing (e.g., apoptosis) of cancer cells inthe event that they develop.

The present invention further provides pharmaceutical compositions(e.g., comprising the cell killing compositions described above). Thepharmaceutical compositions of the present invention may be administeredin a number of ways depending upon whether local or systemic treatmentis desired and upon the area to be treated. Administration may betopical (including ophthalmic and to mucous membranes including vaginaland rectal delivery), pulmonary (e.g., by inhalation or insufflation ofpowders or aerosols, including by nebulizer; intratracheal, intranasal,epidermal and transdermal), oral or parenteral. Parenteraladministration includes intravenous, intraarterial, subcutaneous,intraperitoneal or intramuscular injection or infusion; or intracranial,e.g., intrathecal or intraventricular, administration.

Pharmaceutical compositions and formulations for topical administrationmay include transdermal patches, ointments, lotions, creams, gels,drops, suppositories, sprays, liquids and powders. Conventionalpharmaceutical carriers, aqueous, powder or oily bases, thickeners andthe like may be necessary or desirable.

Compositions and formulations for oral administration include powders orgranules, suspensions or solutions in water or non-aqueous media,capsules, sachets or tablets. Thickeners, flavoring agents, diluents,emulsifiers, dispersing aids or binders may be desirable.

Compositions and formulations for parenteral, intrathecal orintraventricular administration may include sterile aqueous solutionsthat may also contain buffers, diluents and other suitable additivessuch as, but not limited to, penetration enhancers, carrier compoundsand other pharmaceutically acceptable carriers or excipients.

Pharmaceutical compositions of the present invention include, but arenot limited to, solutions, emulsions, and liposome-containingformulations. These compositions may be generated from a variety ofcomponents that include, but are not limited to, preformed liquids,self-emulsifying solids and self-emulsifying semisolids.

The pharmaceutical formulations of the present invention, which mayconveniently be presented in unit dosage form, may be prepared accordingto conventional techniques well known in the pharmaceutical industry.Such techniques include the step of bringing into association the activeingredients with the pharmaceutical carrier(s) or excipient(s). Ingeneral the formulations are prepared by uniformly and intimatelybringing into association the active ingredients with liquid carriers orfinely divided solid carriers or both, and then, if necessary, shapingthe product.

The compositions of the present invention may be formulated into any ofmany possible dosage forms such as, but not limited to, tablets,capsules, liquid syrups, soft gels, suppositories, and enemas. Thecompositions of the present invention may also be formulated assuspensions in aqueous, non-aqueous or mixed media. Aqueous suspensionsmay further contain substances that increase the viscosity of thesuspension including, for example, sodium carboxymethylcellulose,sorbitol and/or dextran. The suspension may also contain stabilizers.

In one embodiment of the present invention the pharmaceuticalcompositions may be formulated and used as foams. Pharmaceutical foamsinclude formulations such as, but not limited to, emulsions,microemulsions, creams, jellies and liposomes. While basically similarin nature these formulations vary in the components and the consistencyof the final product.

The preferred method of administration is by intravenous or IPinjection. It is alternatively possible to use injection into the tumorto be treated. In some embodiments, administration is continued as anadjuvant treatment for an additional period (e.g., several days toseveral months).

The compositions of the present invention may additionally contain otheradjunct components conventionally found in pharmaceutical compositions.Thus, for example, the compositions may contain additional, compatible,pharmaceutically-active materials such as, for example, antipruritics,astringents, local anesthetics or anti-inflammatory agents, or maycontain additional materials useful in physically formulating variousdosage forms of the compositions of the present invention, such as dyes,flavoring agents, preservatives, antioxidants, opacifiers, thickeningagents and stabilizers. However, such materials, when added, should notunduly interfere with the biological activities of the components of thecompositions of the present invention. The formulations can besterilized and, if desired, mixed with auxiliary agents, e.g.,lubricants, preservatives, stabilizers, wetting agents, emulsifiers,salts for influencing osmotic pressure, buffers, colorings, flavoringsand/or aromatic substances and the like which do not deleteriouslyinteract with the active pharmaceutical agents of the formulation.

In some preferred embodiments, a ribonuclease is co-administered withother medical interventions, either simultaneously or sequentially. Forexample, for cancer therapy, any oncolytic agent that is routinely usedin a cancer therapy may be co-administered with the compositions andmethods of the present invention. For example, the U.S. Food and DrugAdministration maintains a formulary of oncolytic agents approved foruse in the United States. International counterpart agencies to theU.S.F.D.A. maintain similar formularies. Table 2 provides a list ofexemplary antineoplastic agents approved for use in the U.S. Thoseskilled in the art will appreciate that the “product labels” required onall U.S. approved chemotherapeutics describe approved indications,dosing information, toxicity data, and the like, for the exemplaryagents. It is contemplated, that in some cases, co-administration withthe compositions of the present invention permits lower doses of suchcompounds, thereby reducing toxicity.

TABLE 2 Aldesleukin (des-alanyl-1, serine- PROLEUKIN Chiron Corp., 125human interleukin-2) Emeryville, CA Alemtuzumab (IgG1κ anti CD52 CAMPATHMillennium and antibody) ILEX Partners, LP, Cambridge, MA Alitretinoin(9-cis-retinoic acid) PANRETIN Ligand Pharmaceuticals, Inc., San DiegoCA Allopurinol (1,5-dihydro-4H- ZYLOPRIM GlaxoSmithKline,pyrazolo[3,4-d]pyrimidin-4-one Research Triangle monosodium salt) Park,NC Altretamine (N,N,N′,N′,N″,N″,- HEXALEN US Bioscience, Westhexamethyl-1,3,5-triazine-2,4,6-triamine) Conshohocken, PA AmifostineETHYOL US Bioscience (ethanethiol, 2-[(3-aminopropyl)amino]-, dihydrogenphosphate (ester)) Anastrozole (1,3- ARIMIDEX AstraZenecaBenzenediacetonitrile,a,a,a′,a′- Pharmaceuticals, LP,tetramethyl-5-(1H-1,2,4-triazol-1- Wilmington, DE ylmethyl)) Arsenictrioxide TRISENOX Cell Therapeutic, Inc., Seattle, WA Asparaginase(L-asparagine amidohydrolase, ELSPAR Merck & Co., Inc., type EC-2)Whitehouse Station, NJ BCG Live (lyophilized preparation of an TICE BCGOrganon Teknika, attenuated strain of Mycobacterium bovis Corp., Durham,NC (Bacillus Calmette-Gukin [BCG], substrain Montreal) BevacizumabAVASTIN Genentech bexarotene capsules (4-[1-(5,6,7,8- TARGRETIN Ligandtetrahydro-3,5,5,8,8-pentamethyl-2- Pharmaceuticals napthalenyl)ethenyl] benzoic acid) bexarotene gel TARGRETIN Ligand PharmaceuticalsBleomycin (cytotoxic glycopeptide antibiotics BLENOXANE Bristol-MyersSquibb produced by Streptomyces verticillus; Co., NY, NY bleomycin A₂and bleomycin B₂) Capecitabine (5′-deoxy-5-fluoro-N- XELODA Roche[(pentyloxy)carbonyl]-cytidine) Carboplatin (platinum, diammine [1,1-PARAPLATIN Bristol-Myers Squibb cyclobutanedicarboxylato(2-)-0,0′]-,(SP-4-2)) Carmustine (1,3-bis(2-chloroethyl)-1- BCNU, BICNUBristol-Myers Squibb nitrosourea) Carmustine with Polifeprosan 20Implant GLIADEL WAFER Guilford Pharmaceuticals, Inc., Baltimore, MDCelecoxib (as 4-[5-(4-methylphenyl)- CELEBREX Searle3-(trifluoromethyl)-1H-pyrazol-1-yl] Pharmaceuticals,benzenesulfonamide) England Cetuximab ERBITUX ImClone/BMS ChlorambucilLEUKERAN GlaxoSmithKline (4-[bis(2chlorethyl)amino]benzenebutanoic acid)Cisplatin (PtCl₂H₆N₂) PLATINOL Bristol-Myers Squibb CladribineLEUSTATIN, 2-CDA R. W. Johnson (2-chloro-2′-deoxy-b-D-adenosine)Pharmaceutical Research Institute, Raritan, NJ Cyclophosphamide(2-[bis(2- CYTOXAN, NEOSAR Bristol-Myers Squibb chloroethyl)amino]tetrahydro-2H- 13,2-oxazaphosphorine 2-oxide monohydrate) Cytarabine(1-b-D-Arabinofuranosylcytosine, CYTOSAR-U Pharmacia & Upjohn C₉H₁₃N₃O₅)Company cytarabine liposomal DEPOCYT Skye Pharmaceuticals, Inc., SanDiego, CA Dacarbazine (5-(3,3-dimethyl-1- DTIC-DOME Bayer AG,triazeno)-imidazole-4-carboxamide (DTIC)) Leverkusen, GermanyDactinomycin, actinomycin D COSMEGEN Merck (actinomycin produced byStreptomyces parvullus, C₆₂H₈₆N₁₂O₁₆) Darbepoetin alfa (recombinantpeptide) ARANESP Amgen, Inc., Thousand Oaks, CA daunorubicin liposomal((8S-cis)-8-acetyl- DANUOXOME Nexstar10-[(3-amino-2,3,6-trideoxy-á-L-lyxo- Pharmaceuticals, Inc.,hexopyranosyl)oxy]-7,8,9,10-tetrahydro- Boulder, CO6,8,11-trihydroxy-1-methoxy-5,12- naphthacenedione hydrochloride)Daunorubicin HCl, daunomycin ((1 S,3 S)- CERUBIDINE Wyeth Ayerst,3-Acetyl-1,2,3,4,6,11-hexahydro-3,5,12- Madison, NJtrihydroxy-10-methoxy-6,11-dioxo-1- naphthacenyl 3-amino-2,3,6-trideoxy-(alpha)-L-lyxo-hexopyranoside hydrochloride) Denileukin diftitox(recombinant peptide) ONTAK Seragen, Inc., Hopkinton, MA Dexrazoxane((S)-4,4′-(1-methyl-1,2- ZINECARD Pharmacia & Upjohnethanediyl)bis-2,6-piperazinedione) Company Docetaxel((2R,3S)-N-carboxy-3- TAXOTERE Aventis phenylisoserine, N-tert-butylester, Pharmaceuticals, Inc., 13-ester with5b-20-epoxy-12a,4,7b,10b,13a- Bridgewater, NJ hexahydroxytax-11-en-9-one4-acetate 2-benzoate, trihydrate) Doxorubicin HCl (8S,10S)-10-[(3-amino-ADRIAMYCIN, Pharmacia & Upjohn2,3,6-trideoxy-a-L-lyxo-hexopyranosyl)oxy]- RUBEX Company8-glycolyl-7,8,9,10-tetrahydro-6,8,11-trihydroxy-1-methoxy-5,12-naphthacenedione hydrochloride) doxorubicinADRIAMYCIN PFS Pharmacia & Upjohn INTRAVENOUS Company INJECTIONdoxorubicin liposomal DOXIL Sequus Pharmaceuticals, Inc., Menlo park, CAdromostanolone propionate (17b-Hydroxy- DROMOSTANOLONE Eli Lilly &Company, 2a-methyl-5a-androstan-3-one propionate) Indianapolis, INDromostanolone propionate MASTERONE Syntex, Corp., Palo INJECTION Alto,CA Elliott's B Solution ELLIOTT'S B Orphan Medical, Inc SOLUTIONEpirubicin ((8S-cis)-10-[(3-amino-2,3,6- ELLENCE Pharmacia & Upjohntrideoxy-a-L-arabino-hexopyranosyl)oxy]- Company7,8,9,10-tetrahydro-6,8,11-trihydroxy-8- (hydroxyacetyl)-1-methoxy-5,12-naphthacenedione hydrochloride) Epoetin alfa (recombinant peptide)EPOGEN Amgen, Inc Erlotinib (N-(3-ethynylphenyl)-6,7-bis(2- TARCEVAGenentech/OSI methoxyethoxy)quinazolin-4-amine) Estramustine(estra-1,3,5(10)-triene-3,17- EMCYT Pharmacia & Upjohn diol(17(beta))-,3-[bis(2- Company chloroethyl)carbamate] 17-(dihydrogen phosphate),disodium salt, monohydrate, or estradiol 3-[bis(2-chloroethyl)carbamate] 17-(dihydrogen phosphate), disodium salt,monohydrate) Etoposide phosphate (4′-Demethyl- ETOPOPHOS Bristol-MyersSquibb epipodophyllotoxin 9-[4,6-O-(R)-ethylidene-(beta)-D-glucopyranoside], 4′-(dihydrogen phosphate)) etoposide, VP-16(4′-demethyl- VEPESID Bristol-Myers Squibb epipodophyllotoxin9-[4,6-0-(R)-ethylidene- (beta)-D-glucopyranoside]) Exemestane(6-methylenandrosta-1,4-diene- AROMASIN Pharmacia & Upjohn 3,17-dione)Company Filgrastim (r-metHuG-CSF) NEUPOGEN Amgen, Inc floxuridine(intraarterial) FUDR Roche (2′-deoxy-5-fluorouridine) Fludarabine(fluorinated nucleotide analog FLUDARA Berlex Laboratories, of theantiviral agent vidarabine, 9-b-D- Inc., Cedar Knolls,arabinofuranosyladenine (ara-A)) NJ Fluorouracil, 5-FU(5-fluoro-2,4(1H,3H)- ADRUCIL ICN Pharmaceuticals, pyrimidinedione)Inc., Humacao, Puerto Rico Fulvestrant (7-alpha-[9-(4,4,5,5,5-pentaFASLODEX IPR Pharmaceuticals, fluoropentylsulphinyl) nonyl]estra-1,3,5-Guayama, Puerto (10)-triene-3,17-beta-diol) Rico Gefitinib(N-(3-chloro-4-fluoro-phenyl)-7- IRESSA AstraZenecamethoxy-6-(3-morpholin-4-ylpropoxy)quinazolin- 4-amine) Gemcitabine(2′-deoxy-2′,2′- GEMZAR Eli Lilly difluorocytidine monohydrochloride(b-isomer)) Gemtuzumab Ozogamicin (anti-CD33 hP67.6) MYLOTARG WyethAyerst Goserelin acetate (acetate salt of ZOLADEX IMPLANT AstraZeneca[D-Ser(But)⁶,Azgly¹⁰]LHRH; Pharmaceuticalspyro-Glu-His-Trp-Ser-Tyr-D-Ser(But)-Leu- Arg-Pro-Azgly-NH2 acetate[C₅₉H₈₄N₁₈O₁₄•(C₂H₄O₂)_(x) Hydroxyurea HYDREA Bristol-Myers SquibbIbritumomab Tiuxetan (immunoconjugate ZEVALIN Biogen IDEC, Inc.,resulting from a thiourea covalent bond Cambridge MA between themonoclonal antibody Ibritumomab and the linker-chelator tiuxetan [N-[2-bis(carboxymethyl)amino]-3-(p- isothiocyanatophenyl)-propyl]-[N-[2-bis(carboxymethyl)amino]-2-(methyl)- ethyl]glycine) Idarubicin(5,12-Naphthacenedione, 9-acetyl- IDAMYCIN Pharmacia & Upjohn7-[(3-amino-2,3,6-trideoxy-(alpha)-L-lyxo- Companyhexopyranosyl)oxy]-7,8,9,10-tetrahydro- 6,9,11-trihydroxyhydrochloride,(7S-cis)) Ifosfamide (3-(2-chloroethyl)-2-[(2- IFEX Bristol-Myers Squibbchloroethyl)amino]tetrahydro-2H-1,3,2- oxazaphosphorine 2-oxide)Imatinib Mesilate (4-[(4-Methyl-1- GLEEVEC Novartis AG, Basel,piperazinyl)methyl]-N-[4-methyl-3-[[4- Switzerland(3-pyridinyl)-2-pyrimidinyl]amino]- phenyl]benzamide methanesulfonate)Interferon alfa-2a (recombinant peptide) ROFERON-A Hoffmann-La Roche,Inc., Nutley, NJ Interferon alfa-2b (recombinant peptide) INTRON ASchering AG, Berlin, (LYOPHILIZED Germany BETASERON) Irinotecan HClCAMPTOSAR Pharmacia & Upjohn ((4S)-4,11-diethyl-4-hydroxy-9-[(4-piperi-Company dinopiperidino)carbonyloxy]-1H-pyrano[3′,4′: 6,7]indolizino[1,2-b] quinoline-3,14(4H,12H) dione hydrochloride trihydrate)Letrozole (4,4′-(1H-1,2,4-Triazol-1- FEMARA Novartis ylmethylene)dibenzonitrile) Leucovorin (L-Glutamic acid, N[4[[(2amino- WELLCOVORIN,Immunex, Corp., 5-formyl-1,4,5,6,7,8 hexahydro4oxo-6- LEUCOVORINSeattle, WA pteridinyl)methyl]amino]benzoyl], calcium salt (1:1))Levamisole HCl ((−)-(S)-2,3,5,6-tetrahydro- ERGAMISOL Janssen Research6-phenylimidazo [2,1-b] thiazole Foundation, monohydrochlorideC₁₁H₁₂N₂S•HCl) Titusville, NJ Lomustine(1-(2-chloro-ethyl)-3-cyclohexyl- CEENU Bristol-Myers Squibb1-nitrosourea) Meclorethamine, nitrogen mustard MUSTARGEN Merck(2-chloro-N-(2-chloroethyl)-N- methylethanamine hydrochloride) Megestrolacetate 17α(acetyloxy)-6- MEGACE Bristol-Myers Squibbmethylpregna-4,6-diene-3,20-dione Melphalan, L-PAM(4-[bis(2-chloroethyl) ALKERAN GlaxoSmithKline amino]-L-phenylalanine)Mercaptopurine, 6-MP (1,7-dihydro-6H- PURINETHOL GlaxoSmithKlinepurine-6-thione monohydrate) Mesna (sodium 2-mercaptoethane sulfonate)MESNEX Asta Medica Methotrexate (N-[4-[[(2,4-diamino-6- METHOTREXATELederle Laboratories pteridinyl)methyl]methylamino]benzoyl]- L-glutamicacid) Methoxsalen (9-methoxy-7H-furo[3,2- UVADEX Therakos, Inc., Wayg][1]-benzopyran-7-one) Exton, Pa Mitomycin C MUTAMYCIN Bristol-MyersSquibb mitomycin C MITOZYTREX SuperGen, Inc., Dublin, CA Mitotane(1,1-dichloro-2-(o-chlorophenyl)- LYSODREN Bristol-Myers Squibb2-(p-chlorophenyl) ethane) Mitoxantrone (1,4-dihydroxy-5,8-bis[[2-NOVANTRONE Immunex [(2-hydroxyethyl)amino]ethyl]amino]-9,10- Corporationanthracenedione dihydrochloride) Nandrolone phenpropionate DURABOLIN-50Organon, Inc., West Orange, NJ Nofetumomab VERLUMA Boehringer IngelheimPharma KG, Germany Oprelvekin (IL-11) NEUMEGA Genetics Institute, Inc.,Alexandria, VA Oxaliplatin (cis-[(1R,2R)-1,2- ELOXATIN SanofiSynthelabo, cyclohexanediamine-N,N′] Inc., NY, NY [oxalato(2-)-O,O′]platinum) Paclitaxel (5β,20-Epoxy-1,2a,4,7β,10β,13a- TAXOL Bristol-MyersSquibb hexahydroxytax-11-en-9-one 4,10-diacetate 2- benzoate 13-esterwith (2R,3S)-N- benzoyl-3-phenylisoserine) Pamidronate (phosphonic acidAREDIA Novartis (3-amino-1-hydroxypropylidene) bis-, disodium salt,pentahydrate, (APD)) Pegademase ((monomethoxypolyethylene ADAGEN Enzonglycol succinimidyl) 11-17-adenosine (PEGADEMASE Pharmaceuticals, Inc.,deaminase) BOVINE) Bridgewater, NJ Pegaspargase (monomethoxypolyethyleneONCASPAR Enzon glycol succinimidyl L-asparaginase) Pegfilgrastim(covalent conjugate of NEULASTA Amgen, Inc recombinant methionyl humanG-CSF (Filgrastim) and monomethoxypolyethylene glycol) PentostatinNIPENT Parke-Davis Pharmaceutical Co., Rockville, MD Pipobroman VERCYTEAbbott Laboratories, Abbott Park, IL Plicamycin, Mithramycin (antibioticMITHRACIN Pfizer, Inc., NY, NY produced by Streptomyces plicatus)Porfimer sodium PHOTOFRIN QLT Phototherapeutics, Inc., Vancouver, CanadaProcarbazine (N-isopropyl-μ-(2- MATULANE Sigma Taumethylhydrazino)-p-toluamide Pharmaceuticals, Inc., monohydrochloride)Gaithersburg, MD Quinacrine (6-chloro-9-(1-methyl-4-diethyl- ATABRINEAbbott Labs amine) butylamino-2-methoxyacridine) Rasburicase(recombinant peptide) ELITEK Sanofi-Synthelabo, Inc., Rituximab(recombinant anti-CD20 RITUXAN Genentech, Inc., antibody) South SanFrancisco, CA Sargramostim (recombinant peptide) PROKINE Immunex CorpSorafenib NEXAVAR Bayer/Onyx Streptozocin (streptozocin 2-deoxy-2-ZANOSAR Pharmacia & Upjohn [[(methylnitrosoamino)carbonyl]amino]-Company a(and b)-D-glucopyranose and 220 mg citric acid anhydrous)Sunitnib malate SUTENT Pfizer Talc (Mg₃SI₄O₁₀ (OH)₂) SCLEROSOL Bryan,Corp., Woburn, MA Tamoxifen ((Z)2-[4-(1,2-diphenyl-1- NOLVADEXAstraZeneca butenyl) phenoxy]-N,N-dimethylethanamine Pharmaceuticals2-hydroxy-1,2,3-propanetricarboxylate (1:1)) Temozolomide(3,4-dihydro-3-methyl-4- TEMODAR Scheringoxoimidazo[5,1-d]-as-tetrazine-8- carboxamide) teniposide, VM-26(4′-demethyl- VUMON Bristol-Myers Squibb epipodophyllotoxin9-[4,6-0-(R)-2- thenylidene-(beta)-D-glucopyranoside]) Testolactone(13-hydroxy-3-oxo-13,17- TESLAC Bristol-Myers Squibbsecoandrosta-1,4-dien-17-oic acid [dgr]-lactone) Thioguanine, 6-TG(2-amino-1,7- THIOGUANINE GlaxoSmithKline dihydro-6H-purine-6-thione)Thiotepa (Aziridine,1,1′,1″- THIOPLEX Immunex phosphinothioylidynetris-,or Tris (1- Corporation aziridinyl) phosphine sulfide) Topotecan HCl((S)-10-[(dimethylamino) HYCAMTIN GlaxoSmithKlinemethyl]-4-ethyl-4,9-dihydroxy-1H- pyrano[3′,4′: 6,7] indolizino [1,2-b]quinoline-3,14-(4H,12H)- dione monohydrochloride) Toremifene(2-(p-[(Z)-4-chloro-1,2- FARESTON Robertsdiphenyl-1-butenyl]-phenoxy)-N,N- Pharmaceutical dimethylethylaminecitrate (1:1)) Corp., Eatontown, NJ Tositumomab, I 131 TositumomabBEXXAR Corixa Corp., Seattle, (recombinant murine immunotherapeutic WAmonoclonal IgG_(2a) lambda anti-CD20 antibody (I 131 is aradioimmunotherapeutic antibody)) Trastuzumab (recombinant monoclonalHERCEPTIN Genentech, Inc IgG₁ kappa anti-HER2 antibody) Tretinoin, ATRA(all-trans retinoic acid) VESANOID Roche Uracil Mustard URACIL MUSTARDRoberts Labs CAPSULES Valrubicin, N-trifluoroacetyladriamycin- VALSTARAnthra --> Medeva 14-valerate ((2S-cis)-2-[1,2,3,4,6,11-hexahydro-2,5,12-trihydroxy-7 methoxy-6,11- dioxo-[[42,3,6-trideoxy-3-[(trifluoroacetyl)-amino-α-L-lyxo-hexopyranosyl]oxyl]-2- naphthacenyl]-2-oxoethylpentanoate) Vinblastine, Leurocristine VELBAN Eli Lilly(C₄₆H₅₆N₄O₁₀•H₂SO₄) Vincristine ONCOVIN Eli Lilly (C₄₆H₅₆N₄O₁₀•H₂SO₄)Vinorelbine (3′,4′-didehydro-4′-deoxy- NAVELBINE GlaxoSmithKlineC′-norvincaleukoblastine [R-(R*,R*)- 2,3-dihydroxybutanedioate(1:2)(salt)]) Zoledronate, Zoledronic acid ((1-Hydroxy- ZOMETA Novartis2-imidazol-1-yl-phosphonoethyl) phosphonic acid monohydrate)

In some embodiments, the RNase of the present invention or a variantthereof is delivered to a target cell using complementation. Forexample, in some embodiments, two or more fragments of RNase aredelivered separately to a cell. The fragments re-associate to form afunctional enzyme. In some embodiments, two protein fragments aredelivered. In other embodiments, vectors comprising nucleic acidsencoding fragments of RNase are introduced into a cell or organismseparately.

Suitable fragments for delivery by complementation may be determined byscreening fragments (e.g., in a cell culture assay) for activity.Preferred fragments are those that rapidly re-associate to form afunctional enzyme. Enzyme activity can be determined using any suitablemethod, including, but not limited to, those disclosed herein.

In some embodiments, the present invention utilizes digestion of RNasesto produce S-peptide and S-protein (See, e.g., Hamachi et al., BioorgMed Chem Lett 9, 1215-1218 (1999); Goldberg and Baldwin, Proc Natl AcadSci, 96, 2019-2024 (1999); Asai et al., J Immun Meth, 299, 63-76 (2005);Backer et al., J Cont Release, 89, 499-511 (2003); Backer et al.,Bioconj Chem, 15, 1021-1029 (2004)). For example, digestion of bovineRNase A by subtilisin results primarily in two fragments due to cleavagebetween Ala20 and Ser21. The shorter fragment (amino acids 1-20) isreferred to as S-peptide, whereas the longer fragment (amino acids20-124) is referred to as S-protein. The two fragments bind tightly atneutral pH and are sometime referred to as RNase S or RNase S′. RNase Sis an active ribonuclease. The S-peptide-5-protein interaction has beenused for affinity purification as well as in tertiary docking systems totarget imaging agents or drugs. Thus, in some embodiments, the presentinvention provides S-peptide-S-protein for human ribonucleases.

In some embodiments, the RNase of the present invention or a variantthereof is provided as a nucleic acid encoding the RNase. Introductionof molecules carrying genetic information into cells is achieved by anyof various methods including, but not limited to, directed injection ofnaked DNA constructs, bombardment with gold particles loaded with saidconstructs, and macromolecule mediated gene transfer using, for example,liposomes, biopolymers, and the like. Preferred methods use genedelivery vehicles derived from viruses, including, but not limited to,adenoviruses, retroviruses, vaccinia viruses, and adeno-associatedviruses. Because of the higher efficiency as compared to retroviruses,vectors derived from adenoviruses are the preferred gene deliveryvehicles for transferring nucleic acid molecules into host cells invivo. Adenoviral vectors have been shown to provide very efficient invivo gene transfer into a variety of solid tumors in animal models andinto human solid tumor xenografts in immune-deficient mice. Examples ofadenoviral vectors and methods for gene transfer are described in PCTpublications WO 00/12738 and WO 00/09675 and U.S. Pat. Appl. Nos.6,033,908, 6,019,978, 6,001,557, 5,994,132, 5,994,128, 5,994,106,5,981,225, 5,885,808, 5,872,154, 5,830,730, and 5,824,544, each of whichis herein incorporated by reference in its entirety.

Vectors may be administered to subject in a variety of ways. Forexample, in some embodiments of the present invention, vectors areadministered into tumors or tissue associated with tumors using directinjection. In other embodiments, administration is via the blood orlymphatic circulation (See e.g., PCT publication 99/02685 hereinincorporated by reference in its entirety). Exemplary dose levels ofadenoviral vector are preferably 10⁸ to 10¹¹ vector particles added tothe perfusate.

Dosing is dependent on severity and responsiveness of the disease stateto be treated, with the course of treatment lasting from several days toseveral months, or until a cure is effected or a diminution of thedisease state is achieved. Optimal dosing schedules can be calculatedfrom measurements of drug accumulation in the body of the patient. Theadministering physician can easily determine optimum dosages, dosingmethodologies and repetition rates. Optimum dosages may vary dependingon the relative potency of individual pharmaceutical compositions, andcan generally be estimated based on EC₅₀s found to be effective in invitro and in vivo animal models or based on the examples describedherein. In general, dosage is from 0.01 μg to 100 g per kg of bodyweight, for example between 0.1 and 1000 mg per kg of body weight,preferably between 0.1 and 500 mg/kg of body weight, and still morepreferably between 0.1 and 200 mg/kg of body weight (e.g., between 1 and100 mg/kg), for a period of between 1 and 240 minutes (e.g., between 2and 60 minutes and preferably between 15 and 45 minutes). Dosages may beadministered as often as need to obtain the desired effect (e.g.,reduction of tumor size or number of cancerous cells), for example onceor more daily to once or more weekly or monthly. In some embodiments,the compositions are administered weekly at a dose of between 0.1 and 10mg (e.g., 1 mg) for a period of between 5 and 60 minutes (e.g., 30minutes). The treating physician can estimate repetition rates fordosing based on measured residence times and concentrations of the drugin bodily fluids or tissues. Following successful treatment, it may bedesirable to have the subject undergo maintenance therapy to prevent therecurrence of the disease state once or more daily, to once every 20years. In some preferred embodiments, dosages are 0.25-1000 mg/kg daily,weekly, or monthly to achieve the desired therapeutic effect. In somepreferred embodiments, dosages are 50 mcg/m² to 400 mcg/m² daily,weekly, or monthly to achieve the desired therapeutic effect. Drugs arealso sometimes dosed in units of activity per dose as opposed to amount(weight) of drug.

All publications, patents, patent applications and sequences identifiedby accession numbers mentioned in the above specification are hereinincorporated by reference in their entirety. Although the invention hasbeen described in connection with specific embodiments, it should beunderstood that the invention as claimed should not be unduly limited tosuch specific embodiments. Modifications and variations of the describedcompositions and methods of the invention that do not significantlychange the functional features of the compositions and methods describedherein are intended to be within the scope of the following claims.

1. A composition comprising a ribonuclease having a human RNase 1sequence with amino acid modification: R4C, G38R, R39G, N67R, G89R,S90R, and V118C.
 2. The composition of claim 1, wherein the ribonucleaseis recombinantly produced.
 3. A method for treating a human subject,comprising administering the composition of claim 1 to a subject,wherein said subject has cancer, is at risk for cancer, or is suspectedof having cancer.
 4. The method of claim 3, wherein the ribonuclease isadministered at 0.01 to 100 mg/kg body weight of the subject per weekfor one or more weeks.
 5. The method of claim 3, wherein theribonuclease is administered at 0.01 to 100 mg/kg body weight of thesubject per day for one or more days.
 6. The method of claim 3, whereinthe ribonuclease is administered at 0.01 to 100 mg/kg body weight of thesubject per treatment for one or more treatments.
 7. The method of claim3, wherein said subject has cancer.
 8. The method of claim 3, whereinsaid subject is at risk for cancer.